The present invention relates to the production of vectors which express alpha-virus genes, such as recombinant viral vectors like adenovirus. The invention further relates to prophylactic and therapeutic vaccines which are protective against these alpha-viruses, such Venezuelan Equine Encephalitis Virus (VEEV), as well as nucleic acids which are used in the vectors, and methods of treatment using the vaccines.
The structural proteins of the alphaviruses are translated from a 26s RNA. The genes encoding these proteins are contained within a single open reading frame in the order:
capsid-E3-E2-6K-E1.
The capsid protein is also known as the xe2x80x9ccorexe2x80x9d and both terms are used herein.
Each protein is either co- or post-translationally cleaved from the poly-protein precursor.
Many prophylactic and therapeutic vaccines rely on the use of recombinant viruses such as vaccinia virus, or adenovirus including replication competant and replication defective adenovirus, for effective delivery of the immunogens.
However, there are sometimes difficulties associated with the expression of alpha viruses in such vectors.
The applicants have found that deletion of a region of an alphavirus gene improves expression in certain vectors and allows expression in other vectors which could otherwise not be made. This is useful in vaccine production.
Thus according to the present invention there is provided a nucleic acid which encodes a polypeptide which produces a protective immune response against an alpha-virus in a mammal to which it is administered, said nucleic acid lacking a competant nuclear targeting signal from a capsid gene thereof.
The nucleic acid as described above may be expressed at enhanced levels, for example in an adenovirus.
As used herein, the term xe2x80x9cpolypeptidexe2x80x9d encompasses short polypeptides as well as proteins. The expression xe2x80x9cenhancedxe2x80x9d means that the expression level of the polypeptide is increased as compared to that which would occur if an otherwise similar nucleic acid including the said portion of the nuclear targeting signal were present.
The nuclear targeting signal (NTS) of any particular alphavirus is either known or it can be determined by alignment with sequences which resemble known nuclear targeting signals in other alphaviruses. For instance, Jakob, Preparative Biochemistry (1995) 25: 99-117 shows the nuclear targeting signal in the Semliki forest virus. An example of a nuclear targeting sequence of VEEV is described below. These sequences are present in the gene encoding the core or capsid protein.
In general, the nuclear targeting signal will be located in a lysine rich area of the genome and will comprise a region which has at least 3 and generally 4 adjacent lysines such as described by Chelsky et al., 1989, Mol+Cell Biology, 9, p2487-2492. In the nucleic acids of the invention, the NTS is inactivated either by complete or partial deletion, or by mutation, for example to alter at least some of the lysine residues.
The nucleic acid of the invention may be a DNA or an RNA molecule, suitably a cDNA. Furthermore it suitably encodes a polypeptide which comprises at least one structural protein of said alpha-virus, and most preferably all of these. Thus, in a preferred embodiment, the recombinant nucleic acid of the invention encodes at least the capsid-E3-E2 proteings of the alpha-virus and more preferably the capsid-E3-E2-6K-E1 proteins of an alpha-virus, provided that the region which encodes the capsid protein lacks a competant nuclear targeting domain.
One alphavirus which has been found to benefit particularly from the present invention is a Venezulan Equine Encephalitis Virus (VEEV). This virus, is a mosquito-borne alphavirus which is an important cause of epidemic disease in humans and of epizootics in horses, donkeys and mules in certain parts of the world, in particular the South Americas.
The existing VEE vaccine, TC-83, was initially produced by attenuation of the Trinidad donkey strain (TRD) of VEE by sequential passage in guinea pig heart cell cultures. However, this vaccine is generally regarded as being inadequate for human vaccination. This is mainly due to the high incidence of side effects in vaccinees and the large proportion of vaccinees who fail to develop neutralising antibodies (Monath et al. 1992, Vaccine Research, 1, 55-68).
A vaccinia-based vaccine against VEE has been constructed (Kinney et al. J. Gen. Virol. 1988, 69, 3005-3013). In this recombinant, 26S RNA encoding structural genes of VEE were inserted into the NYCBH strain of vaccinia. The recombinant virus protected against sub-cutaneous challenge but had limited efficacy against aerosol challenge with VEE.
Attempts to express the full length sequence of this virus in adenovirus, a particularly useful virus for vaccine production, failed completely. However, certain deletion mutants could be successfully expressed. It has been found that the virus proteins will be expressed from a recombinant adenovirus which lacks at least some, and suitably all of nucleotides 7749-7887 within the VEEV genome.
All DNA sequence co-ordinants on the VEEV TC-83 strain followed those of the Trinidad Donkey virus strain (R. M. Kinney et al. Virology (1989) 170, 19-30). The cDNA of the 26s mRNA encoding the TC-83 structural region is that reported by Kinney et al (1988) supra, the content of which is incorporated herein by reference. The virulent Trinidad donkey strain of VEE and the attenuated strain TC-83 have both been cloned and sequenced and the amino acid and nucleotide numbering system used in this reference will be used hereinafter. This work revealed that there are seven amino acid changes between TRD and TC-83. The majority (five) of these changes occur within the gene encoding the glycoprotein E2. The applicants have found a further three changes over and above those described by Kinney as detailed below.
The deletion or omission of a nuclear targeting domain has also been found to improve the expression of other VEEV encoded proteins in other vectors such as plasmids.
Deletion of corresponding regions in other alphaviruses should produce similar enhancements in expression.
The recombinant nucleic acids of the invention may be prepared by any of the well known techniques used in recombinant DNA technology. They may be prepared ab initio using the available chemical methods, for example the automated chemical synthesisers. Alternatively, they may be prepared from wild-type alpha viruses, using known recombinant DNA techniques to generate deletion mutants.
Thus in a further aspect the invention provides a deletion mutant of an alpha-virus, which mutant lacks a nuclear targeting domain, such as a region corresponding to nucleotides 7749-7887 of VEEV. Corresponding regions in other alphaviruses could be readily be determined by comparing sequences and determining analogous regions as is understood in the art. Such comparisons can be made by computer programs.
Confirmation of the nature of the nuclear targeting domain can be confirmed, for example using labelled fragments which may or may not include the purported nuclear targeting domain, and examining where the label appears when the fragments are located in cells transfected with the fragments. The labels may be radiolabels or fluorescent labels as are well known in the art.
Preferably the deletion mutant of the invention is a deletion mutant of VEEV.
A further aspect of the invention provides a recombinant nucleic acid which encodes a deletion mutant of an alpha-virus as described above.
Nucleic acids of the invention are suitably incorporated into vectors, in particular virus vectors like adenovirus (which may be either replication competant or replication defective), vaccinia virus, or in other expression plasmids where they are under the control of a suitable promoter as understood in the art. Preferably the nucleic acids of the invention is incorporated into an adenovirus and most preferably a replication defective adenovirus.
These viruses or plasmids can form vaccines. For this purpose, they will suitably be combined with a pharmaceutically acceptable carrier. Virus vectors are preferably combined with a liquid carrier in an injectable formulation. Plasmid vectors may also be made into an injectable formulation or they may alternatively be bound onto a solid carrier such as a gold bead, which are suitable for administration by means of a gene gun, to the skin of a patient.
In yet a further aspect, the invention provides a method of producing a protective immune response to an alpha-virus, said method comprising administering to a mammal a vaccine as described above. The protective immune response may be used both in prophylaxis and in therapy. Suitable doses will be determined by clinicians taking into account the nature of the patient, the nature of the alphavirus and in the case of therapeutic treatment, as well as the precise nature and form of the vaccine. However in general, when using a virus vector, dosages of the vector will be of at least 104 pfu. For instance, in the case of vaccinia vectors or replication competant adenovirus, dosages are suitably in the range of from 104-1012 pfu (pfu=particle forming units). Replication defective adenovirus may have to be administered at higher dosages.